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WO1999015698A2 - Procede de synthese et d'amplification d'acides nucleiques - Google Patents

Procede de synthese et d'amplification d'acides nucleiques Download PDF

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Publication number
WO1999015698A2
WO1999015698A2 PCT/EP1998/006006 EP9806006W WO9915698A2 WO 1999015698 A2 WO1999015698 A2 WO 1999015698A2 EP 9806006 W EP9806006 W EP 9806006W WO 9915698 A2 WO9915698 A2 WO 9915698A2
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Prior art keywords
nucleic acid
template
initiator
reaction
sequence
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PCT/EP1998/006006
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German (de)
English (en)
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WO1999015698A3 (fr
Inventor
Thomas Ellinger
Ralf Ehricht
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Institut für Molekulare Biotechnologie E.V.
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Application filed by Institut für Molekulare Biotechnologie E.V. filed Critical Institut für Molekulare Biotechnologie E.V.
Priority to EP98951443A priority Critical patent/EP1015640B1/fr
Priority to AT98951443T priority patent/ATE476519T1/de
Priority to AU97452/98A priority patent/AU9745298A/en
Priority to JP2000512986A priority patent/JP2001517458A/ja
Priority to DE59814461T priority patent/DE59814461D1/de
Publication of WO1999015698A2 publication Critical patent/WO1999015698A2/fr
Publication of WO1999015698A3 publication Critical patent/WO1999015698A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions

Definitions

  • the present invention relates to a method for the synthesis and amplification of nucleic acids by means of an enzyme-catalyzed reaction, in which a nucleic acid is copied starting from a single-stranded initiator nucleic acid without the initiator nucleic acid hybridizing to the nucleic acid to be copied.
  • the invention thus relates to a method in which a linear nucleic acid starting from an initiator nucleic acid having a single-stranded region at least at the 3 'end is copied by means of polymerase activity, the initiator nucleic acid not having to have any homology with the nucleic acid to be copied.
  • the reaction on which this method is based is referred to below as initiator-directed amplification (IDA).
  • the first method is based on a DNA polymerase-catalyzed replication of both strands of a target DNA sequence, which is initiated by two oligonucleotide primers homologous to the target DNA sequence becomes.
  • the reaction temperature is varied cyclically to allow denaturation of the DNA template, followed by hybridization of the primers to the target sequence and finally polymerase-catalyzed DNA synthesis.
  • This in vitro amplification technique requires thus, in addition to a special, cyclic temperature regime, the hybridization of specific oligonucleotide primers to a specific DNA template sequence.
  • LCR ligase chain reaction
  • oligonucleotide primers must also be used here, the sequence of which is identical or at least essentially identical to the sequence of template nucleic acid, so that a specific hybridization of the primers to the sequence segments to be amplified can take place.
  • the LCR method by means of which only the sequences represented by the added oligonucleotides can be amplified, has been described in detail, inter alia, in Barany, F. (1991) PCR Methods Appl. 1, 5-16; EP-A2-0 439 182.
  • P & LCR An amplification method (P & LCR) is also known from WO 90/01069, which combines the features of the PCR and the LCR, ie is based on polymerase and ligase-catalyzed reactions.
  • NASBA nucleid acid sequence-based amplification
  • RNA template is degraded by the activity of the RNase H from Escher ichia coli, as a result of which a second primer which comprises the recognition sequence for a viral RNA polymerase, for example T7 RNA polymerase, at its 5 'end, the
  • RNA polymerase synthesizes numerous RNA molecules, the sequence of which corresponds to that of the original target RNA and again serve as a template in subsequent amplification cycles. In this way, the spontaneous switch between reverse transcriptase, RNase H and RNA polymerase catalyzed reactions at a set temperature results in an exponential amplification of the target sequence.
  • SDA Strand displacement amplification
  • an oligonucleotide primer complementary to the target sequence hybridizes to a strand of the target DNA that has been denatured at the beginning.
  • a DNA polymerase preferably the large fragment of DNA polymerase I from E. coli with 5'-exonuclease deficiency (Exo ⁇ Klenow), extends both the primer and the template molecule by copying the 5 'end of the primer to which has a specific restriction site at its 5 'end.
  • RNA-dependent RNA polymerase from the bacteriophage Q ⁇ (Q ⁇ replicase) is used.
  • the RNA probes used in this method contain the sequence elements required for replication by Q ⁇ replicase and an internal segment which is complementary to the target sequence to be detected.
  • the activity of the Q ⁇ replicase copies only those molecules which hybridize with the corresponding target sequence in the added sample. The copies produced then serve in subsequent synthesis steps together with the starting molecule as a template for further replication cycles.
  • Such a method which would thus theoretically enable the synthesis and amplification of any nucleic acid, would be for numerous applications, for example in the fields of molecular biotechnology, in particular genetic engineering in vitro treatment of nucleic acids, evolutionary biotechnology, combinatorial chemistry and molecular information processing invaluable.
  • Such a method could also be used in combination with known primer-dependent amplification methods, especially if specific sequences are to be amplified.
  • the object of the present invention is to provide a method which enables the synthesis and, if appropriate, amplification of nucleic acids without the need for hybridization of complementary nucleic acid molecules to the nucleic acid to be copied.
  • Another object of the invention is to show possible applications for such a hybridization-independent synthesis or amplification technique. These objects are achieved in accordance with the independent claims of the present invention.
  • the subclaims define advantageous embodiments of the invention.
  • the reaction contained two unphosphorylated oligonucleotide primers, each with a length of 20 base pairs, a reverse transcriptase and T7 RNA polymerase.
  • the double-stranded reaction products obtained were distinguished by the fact that they were extended in steps of 20 base pairs.
  • the reaction products were cloned and subjected to a sequence analysis. This sequence analysis showed that in addition to the expected template sequence, these molecules contained several copies of the two primer sequences at their ends as direct repeats.
  • the template and primer sequences and the primer sequences were linked to one another directly, ie without overlap, or showed a deletion or insertion of individual bases at the link.
  • the following amplification Experiments in which only the oligonucleotide primers but no template were added under the same conditions led to the formation of repetitive DNA molecules, the length of which varied in steps of 20 base pairs. They contained a central element of 33 bases in length, which was formed by hybridization of two copies of one of the two primers via the 3 'ends and subsequent filling in to the double strand, as well as a large number of sequences of the two primers fused directly to one another.
  • Polymerase in a template-dependent reaction very effectively extends single-stranded nucleic acid molecules with free 3 '-OH without the single-stranded nucleic acid hybridizing with the template.
  • the sequence of the single strand therefore does not have to be homologous
  • the single strand therefore does not act as a typical primer, but can better be called an initiator.
  • Linear double-stranded DNA serves as a template.
  • the reaction product contains the sequence of the initiator, which is directly fused to the 5 'end of the template. The process is run iteratively and results in longer and longer sequences and a net amplification of the template.
  • the reaction can thus be referred to as initiator-directed amplification ("initiator-directed amplification" or "initiator-dependent amplification", IDA).
  • initiator-directed amplification or "initiator-dependent amplification", IDA).
  • the scheme shows the assumed mechanism of the IDA reaction for the first two reaction cycles.
  • the polymerase simultaneously binds both the end of the double-stranded template and the single-stranded initiator molecule (hatched vertically).
  • the 3 'end of the template is extended so that the initiator is filled up into a double strand.
  • a synthesis takes place into the DNA double strand, which, depending on the binding of polymerase and initiator, can take place either "left-hand side" (black arrows) or "right-hand side” (horizontally hatched arrows).
  • One strand serves as a template, the other is probably replaced as a single strand.
  • the result of the reaction is a DNA double strand in which the sequence of the original double strand template is fused directly with that of the initiator.
  • the single strands created in the "left-hand” and “right-hand” cycle are complementary and can hybridize to form a double strand (gray arrows).
  • a "left-hand” (black arrows) or “right-hand” (horizontally hatched arrows) extension can be carried out.
  • the mechanism is similar to that in the first cycle, but a part of the single strands contains the sequence complementary to the initiator.
  • the IDA reaction according to the invention is isothermal and leads to a net amplification by iterative repetition.
  • An advantageous property of this reaction is above all that it is independent of a specific hybridization, as a result of which any sequence can potentially be fused with any other in the course of the reaction.
  • the IDA reaction enables synthesis on double-stranded template molecules. Thanks to this unique combination of properties, the reaction has great potential for use and opens up completely new possibilities for all areas of genetic engineering in vitro treatment of nucleic acids, evolutionary biotechnology and related tasks.
  • the IDA reaction according to the invention can generally be used for the amplification of nucleic acids.
  • the advantage of the reaction over all known amplification methods is in particular its independence from the structure of the ends of the template. This eliminates the need to create specific primer sequences for each specific amplification problem.
  • pools of nucleic acids with heterologous ends can be amplified in a single reaction using the IDA reaction.
  • the reaction according to the invention can serve as an amplification method alone.
  • the step-by-step extension of the template during the reaction can be carried out by adding a restriction enzyme, the activity of which limits the 5 'end of the
  • RNA initiator molecules in combination with an RNA-degrading enzyme, for example RNAse.
  • the RNA initiator would be at least partially degraded by an RNAse after filling up to the double strand.
  • Nucleic acid analogs for example peptide nucleic acid (PNA; Wittung et al. (1994) Nature 368, 561-563), can also be used as the initiator nucleic acid.
  • reaction according to the invention can also be preceded by a PCR or another primer-dependent amplification reaction.
  • first step the respective template is fused with the initiator sequence. This is followed by a specific amplification in which the initiators can be used as primers.
  • the invented reaction according to the invention can be used for numerous applications which are not covered by already established amplification techniques. Examples include the amplification or copying of total DNA or nucleic acid mixtures, for example for diagnostic purposes or in connection with methods of in vitro evolution (for example, primer-independent amplification in SELEX processes ("systemic evolution of ligands by exponential enrichment"; cf. for example WO 95/30775) or others in vi ro selection processes). It is self-evident that the reaction according to the invention can generally be used in any process in which the duplication of all nucleic acid molecules contained in a sample or generally a random synthesis and
  • Amplification of nucleic acid molecules is desired.
  • single-stranded nucleic acids can also serve as a starting template.
  • an initiator nucleic acid can be used which is complementary to the 3 'end of the single strand to be copied, so that hybridization of the initiator nucleic acid to the template molecule and subsequent polymerase-catalyzed filling of the
  • Doppelstranges a double-stranded template is created, which is then amplified according to the IDA mechanism according to the invention.
  • This method could be used to transcribe RNA into double-stranded DNA with simultaneous amplification and would therefore represent an alternative to RT-PCR (reverse transcriptase-PCR).
  • reaction according to the invention can also be used as the basis for all methods and processes serve that were previously based on copying processes that required a sequence-specific process as a start reaction.
  • methods also include, for example, sequencing and footprint methods, the production of labeled nucleic acids and the production of single-stranded DNA, which can be coupled, for example, with selection methods (for example SELEX processes).
  • the IDA reaction according to the invention can be used to fuse non-complementary nucleic acids. This opens up a number of new opportunities, especially for molecular biotechnology.
  • the method according to the invention allows the production of random sequences from short randomized oligonucleotides and thus the opening up of sequence spaces that were previously practically inaccessible.
  • the IDA method according to the invention is particularly valuable in connection with shuffling technologies (cf. for example Stemmer (1994) Nature 370, 389-391; Stemmer (1994) Proc. Natl. Acad. Sci. USA 91, 10747-10757; WO-Al-95/22625).
  • the IDA method allows the shuffling of DNA fragments without sequence homology. Dosed use in combination with sequence-specific shuffling would allow the global remodeling of biomolecules.
  • This technique can be used for proteins as well as for functional nucleic acids and nucleic acid analogues. This would make it possible on the one hand to optimize multifunctional biomolecules to a desired shape and with suitable selection processes eliminate unwanted functions.
  • different biomolecules with different functions and structures could be fused with one another in such a way that interconnected multifunctional molecules are formed. The result of this new process can be multifunctional proteins, catalytic nucleic acids or aptamers.
  • the IDA reaction takes place in a reaction mixture which contains the following components: an enzyme with polymerase activity, i.e. an enzyme which catalyzes the synthesis of nucleic acid molecules from smaller building blocks, the corresponding building blocks or monomers, usually thus dATP, dCTP, dGTP and dTTP (possibly in modified form), at least one initiator nucleic acid having a single-stranded region at the 3 'end and at least one linear template nucleic acid molecule.
  • an enzyme with polymerase activity i.e. an enzyme which catalyzes the synthesis of nucleic acid molecules from smaller building blocks, the corresponding building blocks or monomers, usually thus dATP, dCTP, dGTP and dTTP (possibly in modified form)
  • at least one initiator nucleic acid having a single-stranded region at the 3 'end and at least one linear template nucleic acid molecule.
  • Template also based on two copies of an initiator molecule.
  • Reverse transcriptase (heterodimer from p66 and p51 subunit, both N-terminally provided with a histidine tag; see Le Grice et al. (1995) Methods Enzymol. 262, 130-144)
  • the enzyme with polymerase activity is used in a concentration of 40 mg / ml to 10 "6 mg / ml. According to a preferred embodiment, the enzyme is used in a final concentration of 4 mg / ml to 10 " 5 mg / ml, whereby a final concentration of 0.4 mg / ml to 0.002 mg / ml is particularly preferred.
  • any single-stranded nucleic acid or any nucleic acid with a single-stranded region located at its 3 'end is used as the initiator.
  • the initiator nucleic acid has a length of at least one nucleotide.
  • the final concentration of the initiator nucleic acid can usually be between 0.2 mM and 2 pM. In a preferred embodiment, it is used in a final concentration of 0.2 mM to 20 nM and particularly preferably in a final concentration of 20 ⁇ M to 2 ⁇ M.
  • the at least one linear template molecule is used according to the invention in a final concentration of 0.2 mM up to a single molecule in a given volume unit.
  • the final concentration of the template is preferably between 5 ⁇ M and 20 pM, particularly preferably between 200 nM and 2 nM.
  • any enzyme with polymerase activity is suitable for use in the IDA reaction.
  • any enzyme with polymerase activity is suitable for use in the IDA reaction.
  • can result from the use of a particular enzyme with polymerase activity can result from the use of a particular enzyme with polymerase activity.
  • the polymerase is a reverse transcriptase or a DNA-dependent DNA polymerase, for example Klenow-Exo " from Escherichia coli (Derbyshire et al. (1988) Science 240, 199-201), particularly preferably an HIV-I reverse transcriptase, and most preferably a histidine tagged HIV-I reverse transcriptase.
  • the IDA reaction is carried out at a temperature between 0 ° C. and 100 ° C., possibly even higher.
  • a reaction temperature between 20 ° C. and 55 ° C. is preferred, particularly preferably a reaction temperature between 30 ° C. and 45 ° C.
  • the reaction temperature is usually at least 0 ° C., preferably at least 50 ° C. The duration of the incubation depends on the desired reaction products, the specific application of the IDA reaction or the desired degree of amplification.
  • spermidine 15 mM MgCl 2 1 mM dNTPs, ie a mixture of 1 mM each of dATP, dGTP, dCTP and dTTP
  • the primers used had the following sequences: 5 '-CCTCTGCAGACTACTATTAC-3' (P1CATCH, unphosphorylated) and
  • HIV-I reverse transcriptase was used as the polymerase, which had previously been prepared using conventional protein purification methods (cf. Protein Purification, Princip. High Res. Meth. And Appl. , Janson and Ryden, VCH Publishers, New York, 1989). This recombinant HIV-I reverse transcriptase as well as its purification and characterization is described in detail in Le Grice et al. (1995) Methods Enzymol. 262, 130-144).
  • Lane 1 shows the reaction mixture with a final concentration of HIV-I reverse transcriptase of 0.4 ⁇ g / ⁇ l, lane 2 with a final concentration of 0.16 ⁇ g / ⁇ l, lane 3 with a final concentration of 0.04 ⁇ g / ⁇ l and lane 4 with a final concentration of 0.008 ⁇ g / ⁇ l.
  • the reaction products were subcloned and individual clones sequenced. This analysis showed that the resulting molecules contained a central element of 33 bases in length, which was formed by self-hybridization of the primer P2CATCH via the 3 'end and subsequent filling in to the double strand, as well as a large number of sequences of the two primers fused together. The initial double strand formation was a necessary prerequisite for the oligomerization of the primers. If the reaction was run with only one primer, the sequence of which excluded self-hybridization, no products were obtained.
  • a double-stranded DNA template of 106 base pairs in length (SP6CATCH) was prepared at a final concentration of 20 nM in a 50 ⁇ l reaction mixture with HIV-I reverse transcriptase (0.4 ⁇ g / ⁇ l final concentration) and a single-stranded initiator with a length of 20 Bases (P1CATCH, 5 ⁇ M final concentration), which was homologous to one of the two 5 'ends of the template, incubated for 2 h at 42 ° C.
  • the reaction mixture also contained:
  • the template and initiator used had the following sequences:
  • reaction products were desalted using Mobispin columns and analyzed using gel electrophoresis (Figure 3). 1/10 of the total reaction mixture was either directly (lane 7) or after treatment with the restriction endonucleases JklcoRI (lane 6), PstI
  • Lane 5 shows 1.5 ⁇ g of a 100 base pair molecular weight ladder (MBI Fermentas, Lithuania). The sizes of the relevant bands are given on the margin.
  • the initial template in an amount of 1 pmol or 0.2 p ol (corresponds to the amount of starting template in lane 4-7).
  • the reaction leads to a distribution of the reaction products over a large molecular weight range above the starting template length (lane 7). Restriction digestion with BcoRI, whose recognition sequence is located near one end of the template, does not remove the heterogeneity of the reaction products, but does reduce their length. Cutting with BcoRI, whose recognition sequence is located near one end of the template, does not remove the heterogeneity of the reaction products, but does reduce their length.
  • PstI whose recognition sequence is located both near the 5 'end of the initiator and near the other end of the template, leads to a dominant specific band which represents a product which is extended by a few bases compared to the starting template.
  • an initiator sequence shortened by 4 bases is retained. Assuming that the initiators were fused directly, ie without overlap with the template sequence, a product length of 118 bases is expected. Cutting the reaction products with PstI and EcoRI removes 4 bases of the template as well as all fused initiators on both sides of the template. The resulting product has an expected length of 98 bases.
  • a double-stranded DNA template with a length of 106 base pairs was at a final concentration of 20 nM in a 50 ⁇ l reaction mixture with HIV-I reverse transcriptase (0.4 ⁇ g / ⁇ l final concentration) and a single-stranded initiator of 35 bases Length (LOOP, 20 ⁇ M final concentration), which was not homologous to either of the 5 'ends of the template, incubated for 2 h at 42 ° C.
  • the reaction mixture also contained: 40 mM Tris pH 7.5 15 mM MgCl 2 16, 6 mM NaCl 1 mM dNTPs, ie a mixture of 1 mM each of dATP, dGTP, dCTP and dTTP
  • BSA bovine serum albumin
  • the template and initiator used had the following sequences:
  • reaction products were desalted using Mobispin columns and analyzed using gel electrophoresis (Figure 4). 1/10 of the total reaction mixture was separated either directly (lane 4) or after treatment with the restriction endonucleases JEcoRI (lane 6), PstI (lane 5) or PstI and EcoRI (lane 7) on a 3% ethidium bromide-stained agarose gel. Lane 1 shows 1.5 ⁇ g of a 100 base pair molecular weight head (MBI Fermentas, Lithuania). The sizes of the relevant bands are given on the margin. In lanes 2 and 3, starting templates were applied in an amount of 2 pmol and 0.2 pmol (corresponds to the amount of starting template in lanes 4-7).
  • the reaction leads to a distribution of the reaction products over a large molecular weight range above the starting template length (lane 4).
  • a double-stranded DNA template of 106 bases in length (SP6CATCH) was prepared at a final concentration of 10 nM in a 50 ⁇ l reaction mixture with different amounts of HIV-I reverse transcriptase and a single-stranded initiator of 35 bases in length (LOOP, 10 ⁇ M Final concentration), which was not homologous to either of the 5 'ends of the template, for 10 min. incubated at 42 ° C.
  • the reaction mixture also contained:
  • dNTPs i.e. a mixture of 200 ⁇ M each of dATP, dGTP, dCTP and dTTP
  • the template and initiator used had the following sequences:
  • Figure 5 shows the reaction products of parallel experiments that differed only in the amount of HIV-I reverse transcriptase added. This was 0.4 ⁇ g / ⁇ l final concentration (lane 2), 0.12 ⁇ g / ⁇ l final concentration (lane 3), 0.04 ⁇ g / ⁇ l final concentration (lane 4), 0.012 ⁇ g / ⁇ l final concentration (lane 5), 0.004 ⁇ g / ⁇ l final concentration (lane 6), 0.0012 ⁇ g / ⁇ l final concentration (lane 7) and no addition of HIV-I reverse transcriptase (lane 8).
  • a fragment is amplified in the course of the PCR, the length of which is the sum of the template length and twice the length corresponds to the initiator (approx. 176 bases).
  • the amplification of this product is particularly efficient when preincubated with a medium concentration of HIV-I reverse transcriptase (lanes 4 to 6).
  • Pre-incubation of the template without HIV-I reverse transcriptase leaves the template unchanged and does not lead to its amplification in the course of the PCR (lane 8).
  • the reaction products were characterized by treatment with restriction endonucleases. Cutting the reaction product with EcoRI or PstI alone, the recognition sequences of which are located at one of the two ends, leads to the removal of one of the two initiators and 4 additional bases of the template and results in a shortening of the reaction product by approx. 39 bases. Cutting with both enzymes leads to the removal of both initiators and 8 additional bases of the template and thus to a shortening of the reaction product by approximately 78 bases.
  • reaction products were cloned and sequenced.
  • sequence analysis confirmed that in the course of the reaction there was a direct, largely overlap-free fusion of template and initiator sequence at both ends of the template, with in some cases an insertion or deletion of fewer bases being detected.

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Abstract

L'invention concerne un procédé de synthèse et d'amplification d'acides nucléiques par réaction à catalyse enzymatique, où un acide nucléique est reproduit sur la base d'un acide nucléique initiateur à un seul brin, sans entraîner d'hybridation de l'acide nucléique initiateur sur l'acide nucléique à reproduire. L'invention concerne également un procédé selon lequel un acide nucléique linéaire est reproduit sur la base d'un acide nucléique initiateur présentant une zone monobrin, du moins à l'extrémité 3', par activité d'une polymérase, l'acide nucléique initiateur n'ayant pas à présenter d'homologie avec l'acide nucléique à reproduire.
PCT/EP1998/006006 1997-09-22 1998-09-21 Procede de synthese et d'amplification d'acides nucleiques WO1999015698A2 (fr)

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Application Number Priority Date Filing Date Title
EP98951443A EP1015640B1 (fr) 1997-09-22 1998-09-21 Procede de synthese et d'amplification d'acides nucleiques
AT98951443T ATE476519T1 (de) 1997-09-22 1998-09-21 Verfahren zur synthese und amplifikation von nukleinsäuren
AU97452/98A AU9745298A (en) 1997-09-22 1998-09-21 Method for synthesising and amplifying nucleic acids
JP2000512986A JP2001517458A (ja) 1997-09-22 1998-09-21 核酸を合成および増幅する方法
DE59814461T DE59814461D1 (de) 1997-09-22 1998-09-21 Verfahren zur synthese und amplifikation von nukleinsäuren

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DE19741714A DE19741714C2 (de) 1997-09-22 1997-09-22 Verfahren zur Synthese und Amplifikation von Nukleinsäuren
DE19741714.0 1997-09-22

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US6994963B1 (en) 2000-07-10 2006-02-07 Ambion, Inc. Methods for recombinatorial nucleic acid synthesis
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US6994963B1 (en) 2000-07-10 2006-02-07 Ambion, Inc. Methods for recombinatorial nucleic acid synthesis
US7910229B2 (en) 2002-05-03 2011-03-22 Ppg Industries Ohio, Inc. Substrate having thermal management coating for an insulating glass unit
DE102004029051B3 (de) * 2004-06-11 2005-07-21 Schunk Gmbh & Co. Kg Fabrik Für Spann- Und Greifwerkzeuge Befestigungseinrichtung zur Befestigung einer Aufsatzbacke an einer beweglich gelagerten Grundbacke eines Linear- oder Zentrischgreifers und Linear- oder Zentrischgreifer
DE102004029051C5 (de) * 2004-06-11 2011-09-15 Schunk Gmbh & Co. Kg Spann- Und Greiftechnik Befestigungseinrichtung zur Befestigung einer Aufsatzbacke an einer beweglich gelagerten Grundbacke eines Linear- oder Zentrischgreifers und Linear- oder Zentrischgreifer

Also Published As

Publication number Publication date
JP2001517458A (ja) 2001-10-09
WO1999015698A3 (fr) 1999-06-10
DE19741714C2 (de) 2002-03-21
AU9745298A (en) 1999-04-12
DE59814461D1 (de) 2010-09-16
EP1015640A2 (fr) 2000-07-05
DE19741714A1 (de) 1999-03-25
EP1015640B1 (fr) 2010-08-04
ATE476519T1 (de) 2010-08-15

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